Herpes Simplex Virus, type 1 (HSV-1) is an enveloped DNA virus that replicates in the nucleus. While HSV is most often found in nature in a latent state in sensory neurons, it efficiently infects and expresses its genome in a very large number of different cell types and tissues. The HSV genome and capsid are also known to accommodate relatively large DNA inserts. Therefore, as a gene delivery vehicle HSV can be used to efficiently vector relatively large or multiple transgenes into a wide range of cell and tissue type. Wild-type HSV is a rapidly growing and very destructive virus, due to activities it encodes that augment or perturb almost every aspect of host cell metabolism. These functions are largely provided by the immediate early (IE) proteins of the virus. A major goal of the previous funding period was to abrogate the expression of HSV IE proteins, provide the means to efficient propagate multiple IE mutants, and evaluate the potential of multiple IE gene knock out viruses as gene delivery vehicles. This systematical undertaking ultimately resulted in the derivation of a whole genome based vector (d109) that; 1. does not express any IE genes, 2. is completely non-toxic to cells, 3. can be obtained in relatively high titers, 4. efficiently delivers its genome to the nucleus, and 5. persists in infected cells for prolonged periods of time. We also found that because this vector does not express any of the HSV activator proteins, transgene expression is fairly low, and that parts of vector genome may integrate into cellular DNA (f= approximately 1%). The goal of this proposal is to complete the development and characterization of the HSV replication-defective vector system for it uses as a general DNA vectoring and expression system. As model transgenes we will use GFP, neo/I, tk and HPRT, and propose 3 specific aims; 1. Methods will be developed to more efficiently insert prospective transgenes and promoters into the vector backbone for evaluation and use. 2. Rational strategies for augmenting or regulating transgene expression in vivo and in vitro will be explored. We will also examine the ability of vector strains of HSV to reactivate previous latent wild type HSV in vivo. 3. The interaction between the vector and cellular genome will be investigated with respect to random insertion, biochemical transformation, and the potential use of HSV as a vector to efficiently deliver genomic DNA for the purpose of promoting homologous recombination between the virus and cell. These latter experiments have implications for the use of HSV to alter cells in an inheritable way, and/or to precisely knock out or repair cellular genes.